Methods and compositions for producing stem cell derived dopaminergic cells for use in treatment of neurodegenerative diseases
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example 1
Materials and Methods
[0124]Neural induction medium (NIM), Y-27632 (10 μM), SB431542 (10 μM) and noggin (100 ng / mL) were combined to create a differentiation medium. Approximately 250 μL of medium was needed per cm2 of differentiation. For patterning to ventral mesencephalic fates, 0.6 to 1.0 μM CHIR99021 and 200 ng / mL Shh-C24II was added to the medium.
[0125]Colonies appeared pluripotent and any differentiated colonies were removed from the culture before initiating differentiation. StemMACS iPSBrew medium was aspirated and cells washed once in PBS. EDTA (0.5 mM) was added to the cells and the cells incubated at room temperature for about 7 minutes. The plate was rocked occasionally to ensure the cells were submerged in EDTA.
[0126]10 mL wash medium (DMEM / F12 with 0.1% albumin or other medium similar to growth medium) was prepared in a 15 mL tube. EDTA was removed and 1 mL wash medium was transferred to the plate.
[0127]Colonies were immediately pipetted off the dish with a pipette and...
example 2
Materials and Methods
[0141]The same materials and protocol disclosed in Example 1 are used for 24 well plates with the following alterations:
[0142]Plates were coated with laminin-111 (1 μg / cm2) in PBS and Ca2+ / Mg2+ (700 μL / cm2). For seeding the cell suspension onto the laminin-111 coated plates, approximately 250 μL was required per cm2 (500 μL / well). To initiate differentiation, for 6 wells of a 24 well plate, 120,000 cells in total were needed.
[0143]At the 96 hour and 168 hour marks after plating, approximately 600 μL medium was added to each well. At the 216 hour mark after plating, approximately 700 μL medium was added to each well.
[0144]Some cell lines required the addition of 10 μM Y-27632 to the medium to survive replating. During replating, approximately 150 μL accutase was added to each well. At the 336 hour mark, approximately 700 μL medium was added per well.
example 3
Preparation of Cells for Transplantation (Day 16-25)
[0145]The same materials and protocol disclosed in Examples 1 and 2 are used.
[0146]Cells were maintained in B27M, BAG (BDNF+AA+GDNF), and FGF8b until the day of transplantation without any further manipulations. Medium was refreshed every 2-3 days. Cells were most suitable for transplantation on day 16 but could be transplanted up until day 26. Cells for transplantation did not receive DAPT or db-cAMP in the medium, as this would result in premature neuronal maturation and increased cell death upon dissociation.
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